Temperature modulates the osmosensitivity of tilapia prolactin cells

In euryhaline fish, prolactin (Prl) plays an essential role in freshwater (FW) acclimation. In the euryhaline and eurythermal Mozambique tilapia, Oreochromis mossambicus, Prl cells are model osmoreceptors, recently described to be thermosensitive. To investigate the effects of temperature on osmoreception, we incubated Prl cells of tilapia acclimated to either FW or seawater (SW) in different combinations of temperatures (20, 26 and 32 °C) and osmolalities (280, 330 and 420 mOsm/kg) for 6 h. Release of both Prl isoforms, Prl188 and Prl177, increased in hyposmotic media and were further augmented with a rise in temperature. Hyposmotically-induced release of Prl188, but not Prl177, was suppressed at 20 °C. In SW fish, mRNA expression of prl188 increased with rising temperatures at lower osmolalities, while and prl177 decreased at 32 °C and higher osmolalities. In Prl cells of SW-acclimated tilapia incubated in hyperosmotic media, the expressions of Prl receptors, prlr1 and prlr2, and the stretch-activated Ca2+ channel, trpv4,decreased at 32 °C, suggesting the presence of a cellular mechanism to compensate for elevated Prl release. Transcription factors, pou1f1, pou2f1b, creb3l1, cebpb, stat3, stat1a and nfat1c, known to regulate prl188 and prl177, were also downregulated at 32 °C. Our findings provide evidence that osmoreception is modulated by temperature, and that both thermal and osmotic responses vary with acclimation salinity.

In vertebrates, hydromineral balance is maintained through osmoregulation.Osmoregulatory processes, in turn, are largely mediated through osmosensitive cells and the neuroendocrine system 1,2 .Euryhaline fishes, which are characterized by their capacity to thrive in a wide range of environmental salinities, have been employed to elucidate the mechanisms underlying the transduction of osmotic stimuli [3][4][5][6][7] .More recently, in light of impending climate-change driven changes in environmental temperature and salinity, a need for cellular and organismal models where the integration of distinct thermal and osmotic stimuli can be studied has emerged 8,9 .The foundational, albeit intricate, nature of endocrine regulation of homeostasis underscores the relevance of understanding the responses of isolated endocrine model systems to combined environmental cues.A first step in understanding the nature of environmental acclimation, therefore, is to characterize acute cellular responses to controlled and physiologically relevant thermal and osmotic stimuli, alone and in combination.
Prolactin (Prl) is a pleiotropic hormone that exerts hundreds of physiological functions in vertebrates including lactation, osmoregulation, growth, reproduction and immune function [10][11][12] .In euryhaline fish, the main function of Prl is to stimulate ion absorption and retention in osmoregulatory tissues to maintain osmotic balance in fresh water (FW) 13,14 .Mozambique tilapia (Oreochromis mossambicus) has been widely used to study the effects of Prl on osmoregulation due to its euryhalinity and the morphology of Prl secreting cells, which comprise a nearly homogeneous portion of the rostral pars distalis (RPD) of the pituitary 15,16 .Consistent with its role in FW adaptation, plasma Prl levels are high in FW and its release increases in pituitaries and dispersed Prl cells incubated in hyposmotic media [17][18][19] .Tilapia Prl cells secrete two isoforms of Prl, Prl 188 and Prl 177 , which are encoded by separate genes 20,21 .Both Prl isoforms act through Prl receptors, Prlr1 and Prlr2, which have been shown to exert distinct downstream effects through JAK/STAT activation and differentially respond to changes in extracellular osmolality 17,22 .Prl 188 responds more robustly to hyposmotic stimuli than Prl 177 17,23 .Due to their importance in FW adaptation, both prl 188 and prl 177 are found to be 10-30 times higher in Prl cells of FW-acclimated tilapia compared with their seawater (SW) counterparts 24,25 .Prl expression, however, is more responsive to hyposmotic stimuli in tilapia acclimated to SW than those in FW 17,26 .Recently, we reported that tilapia Prl cells are also thermosensitive 27 .Insamuch as Mozambique tilapia is both euryhaline and eurythermal, .It remains unclear how thermal stimuli may modulate osmosensitive TFs to regulate prl genes.
Previous studies have shown that low temperature (15 °C) reduces the salinity tolerance of Mozambique tilapia and its hybrids 46,47 .The expression of trpv4, is elevated by temperature in chum salmon (Oncorhynchus keta) 48 and Mozambique tilapia Prl cells 27 , further reinforcing the crosstalk between thermal and osmotic stimuli in the regulation of Prl synthesis and release.While the tilapia Prl cell model has allowed for the identification of several downstream components involved in the transduction of hyposmotic stimuli into Prl secretion, little is known on how temperature interacts with extracellular osmolality in the regulation of prl transcription.For example, it is not known whether temperature can modulate osmotic responses in Prl cells or, conversely, whether extracellular osmolality or acclimation salinity can affect the thermal responses that were recently described 27 .Moreover, the identification of common molecular mechanisms of prl transcription in response to both thermal and osmotic stimuli shall shed light into how Prl cells and other endocrine systems may integrate environmental stimuli with adaptive physiological responses.
In the present study, we employed dispersed Prl cells from SW-and FW-acclimated tilapia in static incubation experiments to investigate Prl 188 and Prl 177 release and transcriptional responses of, prl 188 , prl 177 , prlr1, prlr2, pou1f1, pou2f1b, creb3l1, cebpb, stat3, stat1a, and nfatc1 to changes in osmolality and temperature.This experimental approach allows for the assessment of complex interactions between a fundamental sensory modality, osmoreception, and thermal sensitivity in the endocrine response of a teleost fish model.

Effects of temperature and osmolality on Prl release
The effects of temperature and osmolality on Prl 188 and Prl 177 released from Prl cell incubations of tilapia acclimated to FW and SW by 1 and 6 h are shown in Fig. 1.The patterns of Prl release observed by 6 h were more evident and consistent than those observed by 1 h.In Prl cells of SW-acclimated tilapia, effects of both osmolality and temperature were seen in Prl 188 release by 1 h; hyposmotically-induced Prl 188 release was only observed at 32 °C (Fig. 1A).By 6 h, Prl release was the highest at 32 °C compared with other incubation temperatures in hyposmotic media; hyposmotically-induced Prl release was only observed at 32 °C.In Prl cells of FW-acclimated tilapia, only an osmotic effect was seen by 1 h (Fig. 1B), with hyposmotically-induced Prl 188 release observed at all incubation temperatures.By 6 h, a rise in temperature increased Prl 188 release regardless of incubation osmolality.A five-fold rise in Prl 188 release was seen in cells incubated in hyposmotic media at 32 °C compared with those at 20 °C.Similar to that observed in Prl cells from SW-acclimated fish, hyposmotically-induced Prl 188 release did not occur when Prl cells of FW-acclimated tilapia were incubated at 20 °C.
Prl 177 release was also affected by osmolality by 1 h in SW-acclimated fish, but unlike Prl 188 , no effect of temperature was observed (Fig. 1C); Prl 177 release was inversely related to extracellular osmolaity at 20 and 26 °C.By 6 h, both osmotic and thermal effects were observed in Prl 177 release; Prl 177 release increased with a rise in temperarture and hyposmotically-induced Prl 177 release was observed at all temperatures.
Prl 177 release from Prl cells of FW-acclimated tilapia was affected by both temperature and medium osmolality by 1 and 6 h of incubation (Fig. 1D).By 1 h, hyposmotically-induced Prl 177 release was seen at both 26 °C and 32 °C.By 6 h, hyposmotically-induced Prl 177 release was observed at all temperatures.Similar to SWacclimated fish, Prl 177 release from FW-acclimated fish was decreased at 20 °C compared with 26 and 32 °C, at all temperatures.

Effects of temperature and osmolality on prl mRNA expression
The mRNA expression of prl 188 and prl 177 in tilapia Prl cells incubated for 6 h are shown in Fig. 2. In SW-acclimated tilapia, prl 188 expression was inversely related to media osmolality at all temperatures, and directly related to temperature in isosmotic and hypoosmotic conditions (Fig. 2A).By contrast, in FW-acclimated fish, prl 188 did not vary among treatments (Fig. 2B).In both SW-and FW-acclimated fish, temperature was the only factor affecting prl 177 mRNA expression (Fig. 2 C and D).In SW-fish, prl 177 in isosmotic and hyperosmotic media was higher at 20 °C than at 32 °C, while in FW-fish, prl 177 in hyposmotic and hyperosmotic conditions were higher at 20 °C compared with 26 °C.

Effects of temperature and osmolality on prlr mRNA expression
Main effects of temperature and osmolality were observed in the transcription of prlr1 and prlr2 from Prl cell incubations (Fig. 3).In SW-acclimated tilapia, prlr1 was downregulated at 32 °C compared with other temperatures, while the osmotic effect changed according to temperature (Fig. 3A).In FW-acclimated fish , prlr1 was upregulated as media osmolality increased at all temperatures, while inversely related with temperature in hypo-and hyperosmotic incubations (Fig. 3B).A notable increase of prlr2 (up to ~ threefold) was observed in Prl cells of both SW-and FW-acclimated fish incubated in hyperosmotic media at all temperatures (Fig. 3 C and  D).At 32 °C, expression of prlr2 in Prl cells of SW fish was downregulated at all media osmolalities compared with the other incubation temperatures (Fig. 3 C).

Effects of temperature and osmolality on trpv4 mRNA expression
There were main effects of osmolality and temperature in trpv4 expression in Prl cells of tilapia acclimated to both FW and SW (Fig. 4).In Prl cells of SW-acclimated tilapia, trpv4 expression was higher in hyperosmotic media, with the exception of incubations carried out at 32 °C, where expression was highest in isosmotic conditions (Fig. 4A).In FW-fish, trpv4 expression was increased by rises in extracellular osmolality (Fig. 4B).The expression of trpv4 was inhibited in Prl cells incubated at 20 °C compared with that at 26 °C in fish acclimated to FW, but not those in SW. ).The effects of osmolality and temperature at each time point were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.Grey dashed lines represent baseline control Prl release (330 mOsm/kg:26 °C) after 1 h (0.04 and 0.02 µg/10 5 cells for Prl 188 and Prl 177 released from SW fish, respectively and 0.20 and 0.10 µg/10 5 cells for Prl 188 and Prl 177 released from FW fish, respectively) and grey dotted lines represent baseline Prl release after 6 h (0.08 and 0.06 µg/10 5 cells for Prl 188 and Prl 177 released from SW fish, respectively and 1.04 and 0.41 µg/10 5 cells for Prl 188 and Prl 177 released from FW fish, respectively).

Effects of temperature and osmolality on TF transcript mRNA expression
Main effects of temperature and osmolality were observed in the mRNA levels of most TF transcripts from Prl cell incubations of tilapia acclimated to SW and FW (Figs. 5 and 6).In SW-acclimated tilapia, expression of both pou1f1 (Fig. 5A) and pou2f1b (Fig. 5B) was decreased by high temperature.Expression of pou1f1 was inversely related with media osmolality at both high and low temperatures while there was no osmotic effect on pou2f1b expression.Expression of creb3l1 (Fig. 5C) and cebpb (Fig. 5D) also decreased at high temperature.Both creb3l1 and cebpb were elevated by hyperosmotic media, except for creb3l1 expression at 32 °C.Both stat3 (Fig. 5E) and stat1a (Fig. 5F) were highly expressed at 26 °C; expression in both high and low temperatures was lower than isothermal controls.The expression of stat3 was inversely related with osmolality at all temperatures; stat1a expression was elevated by hyposmotic media only at 32 °C.Similarly, nfatc1 expression was suppressed in hyperosmotic media (Fig. 5G).High temperature inhibited nfatc1 in isosmotic and hyperosmotic media, while both high and low temperatures suppressed hyposmotically-induced nfatc1 expression.
In Prl cells of FW-acclimated tilapia, pou1f1 expression was inversely related with extracellular osmolality at 20 °C and 26 °C (Fig. 6A).A thermal effect on pou1f1 was only seen in isosmotic conditions, where it was downregulated at 32 °C.In isothermal conditions, pou2f1b expression was not affected by extracellular osmolality (Fig. 6B).At 20 °C, pou2f1b was inversely related to osmolality, however, at 32 °C, it increased with osmolality.The expression of pou2f1b in hyposmotic media was inhibited by a rise in temperature, while its expression in hyperosmotic media was elevated at 32 °C.As temperature rose, creb3l1 was downregulated in hyposmotic media (Fig. 6C).There was no temperature effect on cebpb expression; hyperosmotically-induced transcription was observed at all temperatures (Fig. 6D).Hyposmotically-induced stat3 expression was observed at 20 °C and 26 °C, while transcripts in hyposmotic and isosmotic conditions were lowered at 32 °C (Fig. 6E).Medium osmolality did not affect stat1a expression (Fig. 6F); transcription was decreased by low temperature at all media osmolalities.Similarly, nfatc1 was not affected by osmolality, but was inhibited by lower temperatures (Fig. 6G).).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.

Discussion
Stemming from the recent finding that tilapia Prl cells are thermosensitive 27 in addition to their well established role in osmoreception, the present study examined the interactions between osmotic and thermal stimuli in Prl cells of Mozambique tilapia acclimated to either FW or SW.Our findings indicate that: (1) A rise in temperature increases Prl 188 release from dispersed Prl cells from SW-acclimated fish as early as 1 h; (2) The osmotic-sensitivity of Prl 188 release is lost at 20 °C by 6 h; (3) Generally, tilapia acclimated to SW are more responsive to changes in temperature than those acclimated to FW; (4) prlr2 expression in dispersed Prl cells is reduced by a rise in temperature; (5) trpv4 expression in dispersed Prl cells respond differentially to temperature depending on the acclimation salinity of fish; and (6) Most of the TF transcripts in Prl cells of SWacclimated tilapia decrease their mRNA levels in response to an elevation in temperature.
Mozambique tilapia Prl cells are osmoreceptors 7 which have been recently described to also respond to physiologically relevant increases in temperature by increasing Prl release 27 .The control of Prl release by environmental salinity, in vivo, and extracellular osmolality, in vitro, is well studied 17,19,49,50 .Prl cells from FW-acclimated tilapia release more Prl than their SW-counterparts, and respond more robustly to changes in extracellular osmolality 17,26 .As expected, robust hyposmotically-induced Prl 188 release was observed in FW-acclimated tilapia Prl cells, especially by 6 h of incubation; a rise in temperature amplified this effect.In our previous study, both dispersed Prl cells and RPD organoids responded to higher temperatures by elevating Prl 188 release by 6 h of incubation 27 .The present study, however, is the first to test the response of dispersed tilapia Prl cells subjected to 20 °C, which interestingly blocked hyposmotically-induced release of Prl 188 , but not Prl 177 .A previous in-vivo study showed no changes in plasma Prl 188 when tilapia were exposed to temperatures ranging between 20 and 35 °C, though plasma cortisol decreased at higher temperatures 51 .Inasmuch as cortisol has been reported to inhibit Prl release [52][53][54] , the thermally-induced rise in Prl release observed in this and in our previous study 27 , may be further modulated by circulating levels of cortisol in vivo, hence further studies involving both Prl and cortisol are needed to further clarify this response.Moreover, because a rise in temperature also increases Prl cell volume, which mediates Prl release 27 , the observed suppression of hyposmotically-induced Prl release at 20 °C by 6 h may be directly linked to cell volume change.Inasmuch as Prl is pleiotropic, a rise in temperature might affect several other key functions of Prl, including growth and reproduction.In fact, a previous study has shown that warmer water (32 °C) increases growth, while temperatures as low as 22 °C resulted in stunted growth 55 .Moreover, Prl has been linked with testosterone production and gonadal activity of tilapia 56 underscoring the linkage between elevated Prl at high temperatures and increased sexual maturity.While the osmotic sensitivity of Prl 188 release was lost at 20 °C, it did not affect the osmotic responsiveness of Prl 177 release.Prl 177 also exerts somatotropic actions in tilapia 57 and while a reduction in Prl 177 at 20 °C is consistent with lower growth, the retention of its hyposmotic response at that temperature may be vital for FW acclimation in cooler temperatures.The differential responsiveness of Prl 188 and Prl 177 to extracellular osmolality has also been suggested to underlie the observed differences in salinity tolerance between Mozambique tilapia and its congener Nile tilapia, Oreochromis niloticus 58 .Based on the thermal modulation of osmotic responses observed in the present study, it would also be tenable that variations in temperature act in concert with changes in salinity in determining the species-specific environmental regulation of Prl in teleosts.
In FW-acclimated tilapia, prl mRNA did not show any osmosensitivity, consistent with previous studies 17,59 and the notion that prl mRNA levels in FW-fish may be at or near the maximum transcriptional activity and thereby unresponsive to further osmotic stimulation 26 .On the other hand, Prl cells from SW-acclimated tilapia contain low levels of Prls, and therefore, activate prl 177 and prl 188 transcription in hyposmotic conditions 26,37,59 .Accordingly, we observed prl 188 to be responsive to osmotic stimuli in Prl cells of SW-tilapia.Furthermore, in SW-tilapia, prl 177 was not as osmotically sensitive as prl 188 , consistent with previous observations 17 .The two prl ).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.
Vol:.( 1234567890 www.nature.com/scientificreports/transcripts showed opposite expression patterns in response to thermal stimuli.The expression of prl 188 peaked with a rise in temperature in hyposmotic media, indicating that a combination of heat and low osmolality synergizes to maximally induce prl 188 transcription.Thermally-induced Prl release was recently shown to be mediated, at least partially, by a cell-volume dependent mechanism, similar to that involved in hyposmotically-induced Prl release 27 .Consistently, the transcription of prl 188 may be activated in similar fashions by thermal and osmotic stimuli, and further augmented in environments that are both hyposmotic and warm.In Mozambique tilapia, the biological effects of Prls are mediated by Prlr1 and Prlr2, whose transcription in target tissues is also characterized by high osmotic sensitivity 17,60,61 .Expression of both prlrs was affected by temperature and osmolality.Consistent with previous studies 17,22 , the relationship of prlr2 was inversely related to extracellular osmolality in Prl cells of both SW-and FW-acclimated tilapia.Both prlrs were decreased by incubation at 32 °C compared with cooler temperatures (Fig. 3C and D).Regardless of the circulating levels of Prls, the environmental control of their receptors are implicated in modulating the hormonal actions 17,61 .The observed decreases in prlrs with a rise in temperature, especially in Prl cells of SW-acclimated fish incubated in hyperosmotic media, suggests that Prl's effects in high temperature may be attenuated.
The transduction of hyposmotic stimuli in tilapia Prl cells is dependent on the entry of extracellular Ca 2+50,62,63 through trpv4 channels 31,34 .Trpv4 is sensitive to many stimuli including osmotic pressure and heat 64,65 .We observed an increase in trpv4 proportional to that of extracellular osmolality, though this relation was attenuated at the highest incubation temperature.The responses of trpv4 to thermal and osmotic sensitivity differed between Prl cells from FW-and SW-acclimated tilapia, though generally, the transcript was most highly expressed at 26 °C.In our previous study, Prl cells of FW-acclimated tilapia increased trpv4 in response to an elevation in temperature 27 .In the present study, this trend was confirmed, but only when comparing cells incubated at 20 °C and 26 °C.In SW-acclimated tilapia, however, there were no clear effects of thermal regulation of trpv4 expression.Acclimation history plays a vital role in trpv4 expression and it has been reported that Prl cells of SW-acclimated tilapia express four-fold higher trpv4 than their FW counterparts 59 .Hence, a decrease in trpv4 expression observed in Prl cells of SW-acclimated fish incubated at 32 °C may indicate the attenuation of cellular sensitivity to extracellular Ca 2+ entry in response to environmental stimuli, similar to the response of prlrs.It is well accepted that strict regulation of Ca 2+ concentrations in the cytosol is important in Ca 2+ -mediated cell signaling 66 ; a rise in cellular Ca 2+ concentration beyond optimum levels may lead to cytotoxicity and cellular apoptosis 67 .Hence the thermally-induced downregulation of trpv4 in SW-acclimated tilapia could also serve as a protective mechanism to prevent Ca 2+ toxicity.
The transduction of osmotic stimuli into the activation of prl transcription is largely regulated by the activity of TFs and TF modules (TFMs) that operate in the promoter regions of prl 188 and prl 177 genes 29,37 .In Prl cells of tilapia acclimated to SW, expression of TF transcripts was more sensitive to both thermal and osmotic stimuli compared with those in FW.Pit1 and Oct1 have been reported to regulate prl transcription in fish and mammalian models 38,68,69 .In the tilapia RPD, pou1f1 and pou2f1b were the most highly expressed transcripts of Pit and Oct1, respectively 29 .Moreover, pou1f1 expression was inversely related with osmolality in SW-acclimated tilapia 37 .Both pou1f1 and pou2f1b were inhibited by a rise in temperature.Inhibition of these TFs by high temperature reinforces the notion that a compensatory mechanism that attenuates thermally-induced Prl release may also underlie the environmental regulation of tilapia Prl cells.The osmotic sensitivity observed at 32 °C indicates that Prl cells are capable of retaining osmoreceptive functions at higher temperatures.In FW-fish, both pou1f1 and pou2f1b were inversely related to osmolality at 20 °C.At this low temperature, Prl 188 release was reduced relative to higher temperatures and unresponsive to changes in media osmolality; prl 188 expression was unresponsive to both osmotic and thermal stimuli by 6 h.Collectively, these results suggest that, in FW-acclimated tilapia, Prl cells maintained their osmosensitivity through pou1f1 and pou2f1b at 20 °C even though prl 188 mRNA was unchanged across treatments, possibly as a result of pre-existing elevated levels of transcripts and stored Prl 188 .
Hyposmotically-induced Prl release has also been shown to involve the cAMP second messenger system 35,70 .To address downstream changes in this second messenger system, we characterized the response of two transcripts of CREB and CEBP, creb3l1 and cebpb, respectively, which are prevalent in tilapia Prl cells 29 .Similar to the pattern of expression observed for POU genes, a rise in temperature inhibited creb3l1 expression in Prl cells of both SW-and FW-acclimated tilapia.In SW fish, creb3l1 increased in hyperosmotic media at colder temperatures and was attenuated at 32 °C.This expression pattern was quite similar to the expression of trpv4, suggesting a linkage between Ca 2+ and cAMP second messenger systems in the integration of thermal and osmotic responses.By contrast, creb3l1 was not affected by medium osmolality in Prl cells of FW-acclimated tilapia; the only effect observed was the downregulation of the transcript with rising temperature in hyposmotic media.Previously, we reported that raising the temperature from 26 to 32 °C in isosmotic conditions increased trpv4 mRNA expression, but did not affect prl 188 or prl 177 in Prl cells of FW-acclimated tilapia 27 .The downregulation or unresponsiveness of creb3l1 to a rise in temperature may, therefore, contribute to the maintenance of both prls at stable levels at high temperatures.The current results are also consistent with the high expression of creb3l1 reported in SW-tilapia RPDs 29 and the lack of osmotic responsiveness in Prl cells from FW-acclimated fish 37 .Similarly, cebpb followed the expression pattern of trpv4, with upregulation directly proportional to a rise in osmolality.Despite the lack of a thermal effect in Prl cells of FW-acclimated tilapia, cebpb's similarity in response patterns to that of trpv4 during in vitro and in vivo elevations in extracellular osmolality 59 together with its role in encoding an intermediate Ca 2+ binding protein in the cAMP second messenger system 71,72 , reinforces the notion that thermo-and osmosensitive TFs linked to Ca 2+ and cAMP signalling act in concert in the environmental regulation of Prl cells.
Following the binding of Prl to its receptors, Stat proteins mediate the activation of the JAK/STAT signaling pathway 12 .In the present study, stat3 expression in Prl cells of SW-acclimated tilapia was induced in hyposmotic medium at all temperatures.Tilapia Prl cells have been shown to positively respond to both Prl 188 and Prl 177 in vitro, in autocrine fashion 36 .Inasmuch as these autocrine responses occur through Prlrs and the activation of JAK/STAT, understanding the thermal and osmotic modulation of these TFs shall provide further insight into www.nature.com/scientificreports/ the environmental regulation of Prl cells.Both stat3 and stat1a were inhibited at 20 °C and 32 °C, indicating that JAK/STAT signaling is optimized at prevailing ambient temperatures (~ 26 °C).We observed Prl release and prlr expression to have opposite patterns of response to thermal stimuli.The presence of Prl 188 in the medium has been shown to increase Prl release even in hyperosmotic conditions 36 .Therefore, the rise in media Prl concentration at 32 °C might have triggered the reduction of prlr2 and stat3 expression at this warmer temperature, as a long-term negative feedback response.The thermal response of stat1a was similar to that of stat3, although the similarity in osmotic sensitivity was only observed at 32 °C.These results indicate that the responses of stat1a to environmental changes may not be as sensitive as those of stat3, and suggest that during downstream signaling it may be largely sensitive to autocrine regulation by Prls.In Prl cells of FW-acclimated tilapia, stat3 showed similar osmotic sensitivity to their SW counterparts at lower temperatures.At 32 °C, however, osmotic responses were abolished or attenuated in a similar manner as observed with prlr2, suggesting that this receptor isoform and stat3 may be linked during the downstream activation of autocrine signaling.Stat1 is activated by heat in mammalian cell models 73,74 , though downstream signaling effects may differ if Stat1 dimerizes or binds with Stat3 75 .Earlier we found stat3 levels to be similar between RPDs of SW-and FW-acclimated tilapia but stat1a levels were higher in SW fish 29 .Therefore, the distinct patterns we observed in stat transcription may be tied with acclimation salinity.Finally, NFATs have been reported to be activated following rapid Ca 2+ influx 76 and in response to hyperosmotic stress in mammalian cell models and in gills of Atlantic salmon, Salmo salar 44,77,78 .Also, NFAT is reported to form TFMs with AP1, a TF that is sensitive to both hypo-and hyperosmotic stress 76,[79][80][81] .Recently, we reported that the TFM, NFAT_AP1F is activated by both hypo-and hyperosmotic stimuli in tilapia Prl cells 37 .In the present study, nfatc1 expression was reduced in Prl cells of SW-acclimated tilapia by hyperosmotic conditions.The induction of nfatc1 at lower media osmolalities may occur, therefore, in response to hyposmotically-induced Ca 2+ entry.Furthermore, nfatc1 transcription was attenuated by heat.At 32 °C, trpv4 was also inhibited, suggesting that the attenuation of nfatc1 could be linked to a reduction in Ca 2+ influx.At 32 °C, similar patterns of transcription were observed in trpv4, creb3l1, cebpb and nfatc1, underscoring the importance of free Ca 2+ entry to activate prl transcription.In FW fish, nfatc1 expression was reduced at 20 °C and unresponsive to osmotic stimuli.Similarly, trpv4 expression was lower at 20 °C compared with other incubation temperatures.Together, these results are consistent with the notion that extracellular Ca 2+ entry into the intracellular space is important to upregulate nfatc1.
This study unveils the transcriptional responses of molecular regulators involved in prl transcription and Prl release to temperature and extracellular osmolality in a euryhaline and eurythermal fish model that is highly adaptable to environmental fluctuations.Following from our recent finding that Prl cells are thermosensitive 27 , our current results show the extent to which thermally-induced Prl release is modulated by extracellular osmolality; moreover, these responses appeared to be more accentuated in fish acclimated to SW compared with those in FW.In general, at cooler temperatures, Prl 188 release was not as responsive to hyposmotic stimulation as Prl 177 .Rises in temperature further augmented hyposmotically-induced Prl release while at the same time attenuating the transcription of TFs and prlrs involved in the osmoreceptive and autocrine responses of Prl cells, indicating that both temperature and extracellular osmolality modulate Prl cell responses in concert.Even though teleosts are considered ectotherms, these results provide evidence of cellular mechanisms of a pleiotropic endocrine system that sense and respond to unique interactions between thermal and osmotic stimuli.As a result, multiple physiological processes such as growth, development, reproduction and osmoregulation are likely modified following the integrated adaptive responses of Prl cells to changes in environmental temperature and salinity.These findings, therefore, provide novel insights on how fish may be capable of integrating and responding to various environmental cues simultaneously.

Animals
Mature Mozambique tilapia (O.mossambicus) of mixed sexes and sizes (200-1200 g) were obtained from stocks maintained at the Hawai'i Institute of Marine Biology, University of Hawai'i (Kaneohe, HI) and at Mari's Garden (Mililani, HI).Fish were reared in outdoor tanks with a continuous flow of FW or SW at 26 ± 2 °C under natural photoperiod and fed to satiety once a day with trout chow pellets (Skretting, Tooele, UT).Fish were anesthesized with 2-phenoxyethanol (0.3 ml/L, Sigma Aldrich, St. Louis, MO) and euthanized by rapid decapitation prior to sampling.All experimental procedures and methods were conducted in accordance with the ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee, University of Hawai'i.

Experiment 1: Effects of temperature on osmotic sensitivity of FW-acclimated tilapia Prl cells
The effects of environmental temperature on the osmotic sensitivity of FW-acclimated tilapia Prl cells were determined in-vitro by incubating Prl cells at different combinations of media osmolality and temperature.Thirty FW-acclimated Mozambique tilapia of mixed sex weighing 250-1150 g were used.Following euthanasia, RPDs of O. mossambicus were dissected from the pituitary gland and dispersed Prl cells were prepared as previously described 30,36 .Briefly, RPDs were treated with 0.125% (wt/ vol) trypsin (Sigma-Aldrich) dissolved in PBS and placed on a gyratory platform set at 120 rpm for 25 min to allow for complete cell dissociation.The cells were

Figure 1 .
Figure 1.Effects of incubation osmolality and temperature on Prl 188 (A and B) and Prl 177 (C and D) release from Prl cells of SW-acclimated (A and C) and FW-acclimated (B and D) Mozambique tilapia following 1 h and 6 h of incubation.Data are expressed as µg/10 5 cells ± SEM (n = 6-8).The effects of osmolality and temperature at each time point were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.Grey dashed lines represent baseline control Prl release (330 mOsm/kg:26 °C) after 1 h (0.04 and 0.02 µg/10 5 cells for Prl 188 and Prl 177 released from SW fish, respectively and 0.20 and 0.10 µg/10 5 cells for Prl 188 and Prl 177 released from FW fish, respectively) and grey dotted lines represent baseline Prl release after 6 h (0.08 and 0.06 µg/10 5 cells for Prl 188 and Prl 177 released from SW fish, respectively and 1.04 and 0.41 µg/10 5 cells for Prl 188 and Prl 177 released from FW fish, respectively). https://doi.org/10.1038/s41598-023-47044-5www.nature.com/scientificreports/

Figure 2 .
Figure 2. Effects of incubation osmolality and temperature on the mRNA expression of prl 188 and prl 177 in SW-acclimated tilapia (A and C) and FW-acclimated tilapia (B and D) Prl cells after 6 h of incubation.Data are expressed as mean fold change from the isosmotic and isothermal (330 mOsm/kg:26 °C) group ± SEM (n = 6-8).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.

Figure 3 .
Figure 3. Effects of incubation osmolality and temperature on the mRNA expression of prlr1 and prlr2 in SW-acclimated tilapia (A and C) and FW-acclimated tilapia (B and D) Prl cells after 6 h of incubation.Data are expressed as mean fold change from the isosmotic and isothermal (330 mOsm/kg:26 °C) group ± SEM (n = 6-8).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.

Figure 4 .
Figure 4. Effects of incubation osmolality and temperature on the mRNA expression of trpv4 in SW-acclimated tilapia (A) and FW-acclimated tilapia (B) Prl cells after 6 h of incubation.Data are expressed as mean fold change from the isosmotic and isothermal (330 mOsm/kg:26 °C) group ± SEM (n = 6-8).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.

Figure 5 .
Figure 5. Effects of incubation osmolality and temperature on the mRNA expression of pou1f1 (A), pou2f1b (B), creb3l1 (C), cebpb (D), stat3 (E), stat1a (F) and nfatc1 (G) in SW-acclimated tilapia Prl cells after 6 h of incubation.Data are expressed as mean fold change from the isosmotic and isothermal (330 mOsm/kg:26 °C) group ± SEM (n = 6-8).The effects of osmolality and temperature were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).When there was a significant effect of temperature (Temp), media osmolality (Osm) or interaction (Int), group comparisons were conducted using protected Fisher's LSD test.Groups not sharing uppercase letters indicate significant (P < 0.05) mean differences in response to incubation temperatures and groups not sharing lowercase letters reflect significant (P < 0.05) mean differences in response to media osmolality.

Table 1 .
Gene specific primers used for qPCR.